JP6028979B2 - Linker and carrier for nucleic acid solid phase synthesis - Google Patents

Description

  The present invention relates to a linker used for nucleic acid solid-phase synthesis, a carrier for solid-phase synthesis carrying the linker, and a method for producing a nucleic acid using the carrier.

A solid phase synthesis method using a phosphoramidite method is widely used for the chemical synthesis of nucleic acids such as DNA and RNA. In the solid phase phosphoramidite method, nucleic acid synthesis is generally performed by the following steps.
First, a nucleoside that becomes the 3 ′ end of a nucleic acid to be synthesized is ester-bonded with a cleavable linker such as a succinyl group via a 3′-OH group, and is previously supported on a solid phase synthesis carrier (nucleoside linker). Next, the solid phase synthesis carrier carrying the nucleoside linker is placed in a reaction column and set in an automatic nucleic acid synthesizer.
Thereafter, according to the synthesis program of the automatic nucleic acid synthesizer, generally the following steps in the reaction column:
(1) deprotecting the 5′-OH group of the protected nucleoside with an acid such as a trichloroacetic acid / dichloromethane solution;
(2) A step of coupling a nucleoside phosphoramidite (nucleic acid monomer) to a deprotected 5′-OH group in the presence of an activating agent (tetrazole or the like);
(3) A synthetic reaction comprising: capping unreacted 5′-OH group with acetic anhydride and the like; and (4) oxidizing phosphite with hydrous iodine and the like. By repeating this synthesis cycle, the oligonucleotide extension reaction proceeds from the 3 ′ end to the 5 ′ end, whereby a nucleic acid having the target sequence is synthesized.
Finally, the cleavable linker is hydrolyzed with aqueous ammonia or methylamine solution, and the synthesized nucleic acid is separated from the carrier for solid phase synthesis (Non-patent Document 1).

  By the way, when performing the synthesis as described above, as described above, it is necessary to previously support the nucleoside serving as the 3 ′ end as a starting material on a solid phase synthesis carrier via a cleavable linker. In addition, the 3 'end differs depending on the sequence of the nucleic acid to be synthesized. For DNA oligonucleotides, four types of dA, dG, dC, and dT are required, and for RNA, four types of rA, rG, rC, and rU are required. Furthermore, in the case of synthesizing modified oligonucleotides, a solid-phase synthesis carrier that previously supports a modified nucleoside is required, which is complicated.

  Therefore, in order to overcome the above-mentioned problems, a solid-phase synthesis carrier (universal support) carrying a universal linker instead of a nucleoside / succinyl linker that has been generally used as a linker for linking a solid-phase carrier and a starting material. Has been devised. With universal support, no matter what kind of nucleoside or nucleotide is the 3 'end of the nucleic acid to be synthesized, the nucleoside phosphoramidide that becomes the 3' end can be reacted in the same process as normal nucleic acid automatic synthesis. After starting the synthesis and synthesizing the target nucleic acid, it is simply excised from the solid phase synthesis carrier in the same manner as usual. As described above, the solid phase synthesis carrier carrying various nucleoside-linkers is prepared. There is no need.

Several universal supports in which the 3 ′ end of a nucleic acid to be synthesized is a hydroxy group have been proposed (Patent Documents 1 to 4 and Non-Patent Documents 2 and 3). In these universal support structures, there are two adjacent carbon atoms. When one carbon atom has an —OH group as a starting point for nucleic acid synthesis and the other carbon atom has a protective group removed. A group that becomes a nucleophilic group (for example, —OH group, —NH ₂ group, —SH group) is bonded, and when a nucleic acid is cleaved by aqueous ammonia after nucleic acid synthesis, the protecting group of these nucleophilic groups is removed. In this way, the phosphate group is cleaved from the 3 ′ end in a manner that attacks the phosphorus at the 3 ′ end to form a cyclic phosphate. Both are used for the synthesis of nucleic acids whose 3 ′ end is a hydroxy group.
Nucleic acids having a hydroxy group at the 3 ′ end are very useful because they are widely required in the field of biochemistry such as nucleic acid medicine. Under such circumstances, there is a demand for a universal linker capable of synthesizing a nucleic acid having a hydroxy group at the 5 ′ end or 3 ′ end and a universal support carrying the linker.

US Pat. No. 5,681,945 US Pat. No. 6,653,468 International Publication No. 2005/049621 US 2005/0182241

Current Protocols in Nucleic Acid Chemistry (2000) 3.1.1-3.1.28 Bio Techniques, 22, 752-756 (1997) Tetrahedron, 57, 4977-4986 (2001)

An object of the present invention is to provide a universal linker capable of synthesizing a nucleic acid having a hydroxy group at the 3 ′ end, a universal support carrying the linker, and a method for synthesizing a nucleic acid using the universal support.
In particular, the present invention eliminates the need for preparing a solid-phase synthesis carrier that preliminarily supports a modified nucleoside when synthesizing a modified oligonucleotide, and universally has high purity of a nucleic acid having a hydroxy group at the 3 ′ end. An object of the present invention is to provide a solid phase synthesis carrier that can be synthesized by the above method.

That is, the present invention is as follows.
[1] The following general formula

[Where,
X ¹ and X ² each independently represent a protecting group for a hydroxy group that is eliminated by an acid;
L ¹ and L ² each independently represent a linking moiety cleaved by alkali,
R ¹ to R ⁶ are each independently hydrogen _{atom; C 1-6} optionally substituted alkoxy _{C 1-6} alkyl _{group; C 1-6} alkoxy _{group; C 1-7} acyl group; a mono Or a di-C _1-6 alkylamino group; or a halogen atom, or
R ² and R ⁵ together, together with the carbon atom to which they are attached, (1) an oxo group, (2) a C _1-6 alkyl group optionally substituted with a C _1-6 alkoxy group, And (3) a 3- to 8-membered carbocyclic or heterocyclic ring, each of which may be substituted with a substituent selected from a phenyl group which may be substituted with a C _1-6 alkoxy-carbonyl group. ]
A linker for nucleic acid solid phase synthesis, comprising the compound represented by:
[2] The nucleic acid solid phase synthesis linker according to [1], wherein at least one of L ^{1 and} L ² can be linked to a nucleic acid solid phase synthesis carrier.
[3] The linker for nucleic acid solid phase synthesis according to any one of [1] or [2] above, wherein X ¹ and X ² are dimethoxytrityl groups.
[4] The following general formula

[Where,
X ¹ and X ² each independently represent a protecting group for a hydroxy group that is eliminated by an acid;
L ¹ and L ² each independently represent a linking moiety cleaved by alkali,
Sp represents a solid support,
R ¹ to R ⁶ are each independently hydrogen _{atom; C 1-6} optionally substituted alkoxy _{C 1-6} alkyl _{group; C 1-6} alkoxy _{group; C 1-7} acyl group; a mono Or a di-C _1-6 alkylamino group; or a halogen atom, or
R ² and R ⁵ together, together with the carbon atom to which they are attached, (1) an oxo group, (2) a C _1-6 alkyl group optionally substituted with a C _1-6 alkoxy group, And (3) a 3- to 8-membered carbocyclic or heterocyclic ring, each of which may be substituted with a substituent selected from a phenyl group which may be substituted with a C _1-6 alkoxy-carbonyl group. ]
A carrier for solid phase synthesis of nucleic acid having a structure represented by
[5] The nucleic acid solid phase synthesis carrier according to [4], wherein L ¹ is a succinyl group.
[6] The nucleic acid solid phase synthesis carrier according to [4] or [5] above, wherein L ¹ and L ² are succinyl groups.
[7] The carrier for nucleic acid solid phase synthesis according to any one of [4] to [6], wherein X ¹ and X ² are dimethoxytrityl groups.
[8] The nucleic acid solid phase synthesis carrier according to any one of [4] to [7], wherein Sp is a solid phase carrier of porous synthetic polymer particles or porous glass particles.
[9] A method for producing a nucleic acid, comprising a step of performing a nucleic acid synthesis reaction on the nucleic acid solid phase synthesis carrier according to any one of [4] to [8].
[10] The production method of the above-mentioned [9], wherein the nucleic acid synthesis reaction is carried out by a solid phase phosphoramidite method.

The linker for nucleic acid solid phase synthesis of the present invention is a universal linker, and the carrier for nucleic acid synthesis of the present invention carrying the linker is a universal support. The nucleic acid synthesis carrier carrying the linker of the present invention does not require the preparation of a solid phase synthesis carrier carrying the nucleotide that becomes the 3 ′ end of the nucleic acid to be synthesized, and a hydroxy group is introduced at the 3 ′ end. The synthesized nucleic acid can be synthesized with high purity. When synthesizing modified oligonucleotides, it is not necessary to prepare a carrier for solid-phase synthesis that pre-supports modified nucleosides or nucleotides, and nucleic acids with a hydroxy group introduced at the 3 'end are synthesized with high purity. can do.
Therefore, the carrier for nucleic acid synthesis of the present invention is suitably used for automatic synthesis of a nucleic acid having a hydroxy group at the 3 ′ end. Furthermore, the method for producing a nucleic acid using the carrier for nucleic acid synthesis of the present invention does not require a separate hydroxy group to be introduced at the 3 ′ end of the nucleic acid synthesized as in the conventional method.

FIG. 1 is a diagram showing an HPLC chart of the DNA oligonucleotide solution obtained in Example 2 (a) and Comparative Example 1 (b). FIG. 2 is an HPLC chart of the DNA oligonucleotide solutions obtained in Example 4 (a) and Comparative Example 2 (b). FIG. 3 is a diagram showing an HPLC chart of the DNA oligonucleotide solutions obtained in Example 6 (a) and Comparative Example 3 (b).

  In the present specification, the “nucleic acid” means a chain compound (oligonucleotide) in which nucleotides are linked by a phosphodiester bond, and includes DNA, RNA and the like. The nucleic acid may be either single-stranded or double-stranded, but is preferably single-stranded because efficient synthesis by a nucleic acid synthesizer is possible. As used herein, the term “nucleic acid” includes not only oligonucleotides containing purine bases such as adenine (A) and guanine (G) and pyrimidine bases such as thymine (T), cytosine (C) and uracil (U). Also included are modified oligonucleotides containing other modified heterocyclic bases.

  The nucleotide length of the nucleic acid is not particularly limited, but is preferably 2 to 200 nucleotides. This is because when the nucleotide length is too long, the yield and purity of the nucleic acid obtained are lowered.

  As used herein, “linker” refers to a molecule that connects two substances through a covalent bond. In the present invention, the linker links the solid phase carrier and the nucleic acid.

  The linker for solid-phase nucleic acid synthesis of the present invention is represented by the following general formula.

Where
X ¹ and X ² each independently represent a protecting group for a hydroxy group that is eliminated by an acid.
Examples of the protecting group include a trityl group (Tr), a monomethoxytrityl group (MMTr), a dimethoxytrityl group (DMTr), and the like. These protecting groups can be removed using a solution of Bronsted acid such as trichloroacetic acid or dichloroacetic acid in dichloromethane or toluene. Since deprotection with acid is easy, ^{X 1} and ^{X 2} DMTr is preferred.

L ¹ and L ² each independently represent a linking moiety cleaved by alkali, and it is sufficient that at least one of L ¹ and L ² can be linked to a nucleic acid solid phase synthesis carrier. Examples of L ¹ and L ² include, but are not limited to, a succinyl group (succinyl linker), an acetyl group, or a Q linker (Pon et al., Nucleic Acids Res., 27, 1531 (1999)). In order to minimize the number of steps of the linker synthesis, cheaper cost, preferably at least one of L ¹ and L ² are succinyl groups, more preferably L ¹ and L ² are succinyl groups. These linking portions can be easily hydrolyzed and cleaved with aqueous ammonia or aqueous ammonia / methylamine mixture, and the oligonucleotide is cleaved from the solid phase synthesis carrier after completion of the automatic synthesis. That is, L ¹ and L ² were cleaved from the linker for nucleic acid solid phase synthesis of the present invention represented by the above formula, and a molecule in which oligonucleotides synthesized at positions X ¹ and X ² were bound was generated at the same time. A synthetic oligonucleotide having a hydroxy group at the 3 ′ end, in which the linker of the present invention is released from the synthetic oligonucleotide in such a manner that a phosphoric acid ester is generated by attacking the phosphorus atom at the 3 ′ end by a nucleophilic reaction of “O ⁻ ” Can be obtained. The reaction as the universal support of the present invention will be described later.

R ¹ to R ⁶ are each independently hydrogen _{atom; C 1-6} optionally substituted alkoxy _{C 1-6} alkyl _{group; C 1-6} alkoxy _{group; C 1-7} acyl group; a mono Or a di-C _1-6 alkylamino group; or a halogen atom, or
R ² and R ⁵ together, together with the carbon atom to which they are attached, (1) an oxo group, (2) a C _1-6 alkyl group optionally substituted with a C _1-6 alkoxy group, And (3) a 3- to 8-membered carbocyclic or heterocyclic ring, each of which may be substituted with a substituent selected from a phenyl group which may be substituted with a C _1-6 alkoxy-carbonyl group.

In the present specification, as the “C _1-6 alkyl group”, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n- Examples thereof include an amyl group, an isoamyl group, a sec-amyl group, a tert-amyl group, and a hexyl group. Of these, a methyl group or an ethyl group is preferable.

In the present specification, the “C _1-6 alkoxy group” includes a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, and pentyl. An oxy group, a hexyloxy group, etc. are mentioned. Of these, a methoxy group is preferred.

In the present specification, the “C _1-6 alkyl group optionally substituted with a C _1-6 alkoxy group” includes, in addition to the examples in the above “C _1-6 alkyl group”, a methoxymethyl group, methoxy Examples thereof include an ethyl group, a methoxy n-propyl group, a methoxyisopropyl group, an ethoxymethyl group, an n-propoxymethyl group, and an n-butoxymethyl group. Of these, a methyl group, an ethyl group, a methoxymethyl group or a methoxyethyl group is preferable.

In the present specification, the “C _1-7 acyl group”
(1) formyl group,
(2) a carboxy group,
(3) a C _1-6 alkyl-carbonyl group,
(4) a C _1-6 alkoxy-carbonyl group,
Etc.
Examples of the “C _1-6 alkyl-carbonyl group” include acetyl group, propanoyl group, butanoyl group, isobutanoyl group, pentanoyl group, isopentanoyl group, hexanoyl group and the like. Of these, an acetyl group is preferred.
Examples of the “C _1-6 alkoxy-carbonyl group” include methoxycarbonyl group, ethoxycarbonyl group, n-propoxycarbonyl group, isopropoxycarbonyl group, n-butoxycarbonyl group, pentyloxycarbonyl group, hexyloxycarbonyl group and the like. It is done. Of these, a methoxycarbonyl group is preferred.
Among the above-mentioned “C _1-7 acyl group”, a C _1-6 alkoxy-carbonyl group is preferable, and a methoxycarbonyl group is particularly preferable.

In the present specification, the “mono or di-C _1-6 alkylamino group” includes a methylamino group, ethylamino group, n-propylamino group, isopropylamino group, n-butylamino group, isobutylamino group, sec -Butylamino group, tert-butylamino group, n-amylamino group, isoamylamino group, sec-amylamino group, tert-amylamino group, hexylamino group, dimethylamino group, diethylamino group, di-n-propylamino group, diisopropylamino Group, di-n-butylamino group and the like. Of these, a methylamino group, a dimethylamino group, an ethylamino group or a diethylamino group is preferable.

  In the present specification, examples of the “halogen atom” include a chlorine atom, a fluorine atom, a bromine atom, and an iodine atom. Of these, a chlorine atom or a fluorine atom is preferred.

In the present specification, as the “3- to 8-membered carbocycle”, for example,
C _3-8 cycloalkanes such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane;
C _3-8 cycloalkene such as cyclopropene, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclooctene;
Is mentioned. Among these, C _5-6 cycloalkane or C _5-6 cycloalkene is preferable.

In the present specification, the “3- to 8-membered heterocyclic ring” includes, for example, 3 to 8 containing 1 to 4 heteroatoms selected from an oxygen atom, a sulfur atom and a nitrogen atom in addition to a carbon atom as a ring constituent atom. Examples include 5-membered (preferably 5- or 6-membered) aromatic heterocyclic groups and non-aromatic heterocyclic groups such as furan, thiophene, pyridine, pyrimidine, pyridazine, pyrazine, pyrrole, imidazole, pyrazole, thiazole, and isothiazole. 3- to 8-membered aromatic heterocycles such as oxazole, isoxazole, oxadiazole, thiadiazole, triazole, triazine;
Aziridine, azetidine, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, hexamethyleneimine, oxazolidine, thiazolidine, imidazolidine, oxazoline, thiazoline, imidazoline, dioxol, dioxolane, dihydrooxadiazole, pyran, dihydropyran, tetrahydropyran, thiopyran 3 to 8 membered non-aromatic heterocycles such as, tetrahydrothiopyran, dihydrofuran, tetrahydrofuran, pyrazolidine, pyrazoline, dihydropyridine, tetrahydropyridine, dihydropyrimidine, tetrahydropyrimidine, dihydrotriazole, tetrahydrotriazole;
Etc. Of these, a 5- or 6-membered non-aromatic heterocyclic ring (preferably tetrahydrofuran or pyrrolidine) is preferable.

In the present specification, the “phenyl group optionally substituted with a C _1-6 alkoxy-carbonyl group” includes a phenyl group, a 2-methoxycarbonylphenyl group, a 3-methoxycarbonylphenyl group, and a 4-methoxycarbonylphenyl group. 2-ethoxycarbonylphenyl group, 3-ethoxycarbonylphenyl group, 4-ethoxycarbonylphenyl group, 2-n-propoxycarbonylphenyl group, 3-n-propoxycarbonylphenyl group, 4-n-propoxycarbonylphenyl group, 2 -Isopropoxycarbonylphenyl group, 3-isopropoxycarbonylphenyl group, 4-isopropoxycarbonylphenyl group, etc. are mentioned. Among them, phenyl group, 2-methoxycarbonylphenyl group, 3-methoxycarbonylphenyl group, 4-methoxycarbonylphenyl group, 2-ethoxycarbonylphenyl group, 3-ethoxycarbonylphenyl group, 4-ethoxycarbonylphenyl group, 2-ethoxy A carbonylphenyl group, a 3-ethoxycarbonylphenyl group or a 4-ethoxycarbonylphenyl group is preferred.

R ¹ is preferably a hydrogen atom.
R ³ is preferably a hydrogen atom.
R ⁴ is preferably a hydrogen atom.
R ⁶ is preferably a hydrogen atom.
R ² is preferably a C _1-7 acyl group, more preferably a C _1-6 alkoxy-carbonyl group, and particularly preferably a methoxycarbonyl group.
R ⁵ is preferably a C _1-7 acyl group, more preferably a C _1-6 alkoxy-carbonyl group, and particularly preferably a methoxycarbonyl group.
Alternatively, R ² and R ⁵ together with the carbon atom to which they are attached, (1) an oxo group, (2) a C _1-6 alkyl group substituted with a C _1-6 alkoxy group, and (3) forming a 5- or 6-membered non-aromatic heterocyclic ring (preferably tetrahydrofuran or pyrrolidine), each of which may be substituted with a substituent selected from a phenyl group substituted with a C _1-6 alkoxy-carbonyl group To do.

  The solid phase carrier for nucleic acid solid phase synthesis of the present invention carries the nucleic acid solid phase synthesis linker of the present invention and is represented by the following general formula.

In the formula, X ¹ and X ² , L ¹ and L ² , and R ^{1 to} R ⁶ are as defined above.

In the formula, Sp represents a solid phase carrier. For convenience, Sp is bonded to L ¹ , but may be bonded to L ² . The solid phase carrier is not particularly limited as long as it can easily remove the excessively used reagent by washing. For example, a porous synthetic polymer carrier such as a glass-based porous carrier, a polystyrene-based carrier, or an acrylamide-based carrier can be used. Can be mentioned.

In a preferred embodiment of the present invention, in the above formula, Sp is a glass-based porous carrier.
Here, the “glass-based porous carrier” refers to a porous carrier containing glass as a constituent component, and examples thereof include, but are not limited to, particle-shaped porous glass particles (CPG). More specifically, as the CPG, a CPG solid phase carrier having a long chain aminoalkyl spacer (LCAA-CPG solid phase carrier) is preferably used. Further, in the case of synthesis of a long chain nucleotide, Most preferably, the CPG pores are 20 to 400 nm, more preferably 50 to 200 nm, still more preferably 100 nm.

  In another preferred embodiment of the present invention, in the above formula, Sp is a polystyrene-based support. Here, the “polystyrene carrier” means the following formula:

The solid-phase support | carrier comprised from the copolymer containing the structural unit (A) represented by these, and / or its substituted as a structural unit. In the substitution product of the structural unit (A), one or more hydrogen atoms (including a hydrogen atom of a benzene ring) contained in the structural unit (A) are substituted with an alkyl group having 1 to 6 carbon atoms (for example, a methyl group, Ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-amyl group, isoamyl group, sec-amyl group, tert-amyl group), halogen atom , Amino groups, hydroxy groups, carboxyl groups, sulfone groups, cyano groups, methoxy groups, nitro groups, vinyl groups and the like. The substituent is preferably an amino group or a hydroxy group. The position of the substituent is not particularly limited, but is preferably para to the main chain on the benzene ring. As a preferable substituent of the structural unit (A), there can be mentioned a hydroxystyrene structural unit (B) represented by the following formula.

  The amount of the structural unit (A) and / or its substitution product with respect to the total amount of structural units contained in the copolymer constituting the polystyrene carrier is not particularly limited, but is usually 50 to 100% by weight, preferably 60 to 100%. % By weight.

  Specific examples of the monomer compound copolymerizable with the structural unit (A) include styrene; o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, ethylstyrene, and trimethylstyrene. And nuclear alkyl-substituted styrenes such as pt-butylstyrene; α-alkyl-substituted styrenes such as α-methylstyrene and α-methyl-p-methylstyrene; chlorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, And halogenated styrene such as bromostyrene; halogenated alkylstyrene such as chloromethylstyrene and fluoromethylstyrene; hydroxystyrene; hydroxymethylstyrene; vinyl benzoate; sodium styrenesulfonate; cyanostyrene; Ethoxystyrene; butoxystyrene; nitrostyrene; acetoxystyrene, and acyloxystyrene etc. benzoxycarbonyl and styrene, but not limited to. Furthermore, (meth) acrylic acid alkyl ester monomers such as methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, etc., acrylonitrile, methacrylonitrile, eta Examples thereof include, but are not limited to, vinyl cyanide monomers such as acrylonitrile.

  In another embodiment of the present invention, in the above formula, Sp represents the following formula in addition to the structural units (A) and (B):

A solid phase carrier comprising styrene-hydroxystyrene-divinylbenzene copolymer particles further having a divinylbenzene structural unit (C) represented by the formula (JP 2005-097545 A, JP 2005-325272 A and JP 2006-342245 A). ), Or a solid phase carrier made of a styrene- (meth) acrylonitrile-hydroxystyrene-divinylbenzene copolymer (Japanese Patent Laid-Open No. 2008-074979). In addition, one or more hydrogen atoms (including hydrogen atoms of a benzene ring) in the divinylbenzene structural unit (C) are an alkyl group having 1 to 6 carbon atoms (for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group). Group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-amyl group, isoamyl group, sec-amyl group, tert-amyl group), halogen atom, amino group, hydroxy group, carboxyl group , A sulfone group, a cyano group, a methoxy group, a nitro group, a vinyl group and the like may be substituted. The substituent is preferably an amino group or a hydroxy group.

  In the above formula, when Sp is a solid phase carrier comprising the styrene- (meth) acrylonitrile-hydroxystyrene-divinylbenzene copolymer, the structural unit of (meth) acrylonitrile is each of acrylonitrile or methacrylonitrile. Each structural unit may be included alone or both. The amount of structural units of (meth) acrylonitrile relative to the total amount of structural units in the styrene- (meth) acrylonitrile-hydroxystyrene-divinylbenzene copolymer may be determined depending on the type of organic solvent, whether it is too much or too little. Fluctuation increases. Therefore, it is preferably 2 to 11 mmol / g.

  In another preferred embodiment of the present invention, in the above formula, Sp is an acrylamide-based carrier. More specifically, in the above formula, Sp is an aromatic monovinyl compound-divinyl compound- (meth) acrylamide derivative system further containing a (meth) acrylamide derivative monomer in addition to the structural units (A) and (C). It may be a solid phase synthesis carrier made of a copolymer.

  In the above formula, when Sp is a solid phase carrier comprising the aromatic monovinyl compound-divinyl compound- (meth) acrylamide derivative copolymer, the aromatic monovinyl compound is not particularly limited, Styrene; Nuclear alkyl-substituted styrenes such as o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, ethylstyrene, trimethylstyrene, and pt-butylstyrene; α-methylstyrene, and α-alkyl-substituted styrenes such as α-methyl-p-methylstyrene; nuclear halogenated styrenes such as chlorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, and bromostyrene; halogens such as chloromethylstyrene and fluoromethylstyrene Alkyls Hydroxy styrene; hydroxymethyl styrene; vinyl benzoate; sodium styrene sulfonate; cyano styrene; methoxy styrene; ethoxy styrene; butoxy styrene; nitro styrene; acyloxy styrene such as acetoxy styrene and benzoxy styrene; Is styrene.

  Examples of the (meth) acrylamide derivative include N-alkyl (meth) acrylamide; N, N-dialkyl (meth) acrylamide; N-alkoxyalkyl (meth) acrylamide; 2- (meth) acrylamide alkanesulfonic acid; N-alkylol (meth) acrylamide such as methylol (meth) acrylamide; acrylamide; methacrylamide; diacetone acrylamide; N, N-dimethylaminopropyl acrylamide; acroylmorpholine; N-phenoxymethyl (meth) acrylamide However, it is not limited to these.

  The alkyl contained in the N-alkyl (meth) acrylamide is a linear or branched alkyl having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. As N-alkyl (meth) acrylamide, N-methyl (meth) acrylamide, N-ethyl (meth) acrylamide, Nn-propyl (meth) acrylamide, N-isopropyl (meth) acrylamide, N-tert-butyl ( Examples include meth) acrylamide and N-lauryl (meth) acrylamide.

The two alkyls contained in the N, N-dialkyl (meth) acrylamide are each a linear or branched alkyl having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. As N, N-dialkyl (meth) acrylamide, N, N-dimethyl (meth) acrylamide, N, N-diethyl (meth) acrylamide, N, N-diisopropyl (meth) acrylamide, N, N-di-tert- Butyl (meth) acrylamide, N, N-dilauryl (meth) acrylamide, N, N-di-tert-octyl (meth) acrylamide, N, N-dilauryl (meth) acrylamide, and N, N-dicyclohexyl (meth) acrylamide Etc. can be mentioned.

  The alkoxy contained in the N-alkoxyalkyl (meth) acrylamide is a linear or branched alkoxy having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. The alkyl contained in N-alkoxyalkyl (meth) acrylamide is a linear or branched alkyl having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. As N-alkoxyalkyl (meth) acrylamide, N-methoxymethyl (meth) acrylamide, N-ethoxymethyl (meth) acrylamide, N-propoxymethyl (meth) acrylamide, N-butoxymethyl (meth) acrylamide, N-ethoxy Examples thereof include ethyl (meth) acrylamide, N-methoxypropyl (meth) acrylamide, N-ethoxypropyl (meth) acrylamide, and N-isopropoxyethyl (meth) acrylamide.

  The alkane contained in the 2- (meth) acrylamide alkanesulfonic acid is usually a linear or branched alkane having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. As 2- (meth) acrylamide alkanesulfonic acid, 2-acrylamidopropanesulfonic acid, 2-acrylamido-n-butanesulfonic acid, 2-acrylamido-n-hexanesulfonic acid, and 2-acrylamido-2-methylpropanesulfonic acid Etc. can be mentioned.

  The (meth) acrylamide derivative is preferably diacetone acrylamide, N-isopropyl (meth) acrylamide, N-methoxymethyl (meth) acrylamide or N, N-dimethyl (meth) acrylamide.

  Examples of the structural unit derived from a typical (meth) acrylamide derivative monomer include the following structural units.

  In the above formula, when Sp is an acrylamide-based solid phase carrier, if the content of the structural unit derived from the (meth) acrylamide derivative monomer is too small, a decrease in the synthesis amount of nucleic acid and a decrease in the synthesis purity can be avoided. On the other hand, if it is too much, it is difficult to form porous resin beads. Therefore, it is preferably 0.3 to 4 mmol / g, more preferably 0.4 to 3.5 mmol / g, and still more preferably 0.6 to 3 mmol / g.

The porous particles used as the solid phase synthesis carrier of the present invention preferably have a functional group that contributes to nucleic acid synthesis. “Contributing to nucleic acid synthesis” is a functional group capable of adding a linker, which can be a starting point of nucleic acid synthesis, and specifically includes an amino group, a hydroxy group, and the like.
The content of the functional group that contributes to nucleic acid synthesis is not particularly limited, but if the content of the functional group is too small, the yield of nucleic acid is reduced, while the content of the functional group is too large. The purity of the obtained nucleic acid is reduced. Therefore, it is preferably 10 to 2000 μmol / g, more preferably 50 to 1000 μmol / g, and still more preferably 100 to 800 μmol / g.

When the functional group contributing to nucleic acid synthesis is a hydroxy group, the amount of the hydroxy group of the porous particle of the present invention is measured by titration based on JIS K0070. Specifically, pyridine is prepared by adding pyridine to 25 g of acetic anhydride so that the total amount becomes 100 mL. 0.5 mL of the acetylating reagent, 4.5 mL of pyridine, and about 0.5 g of a sample are placed in a flask and heated at 95-100 ° C. for 2 hours to acetylate the hydroxy group. Next, 1 mL of distilled water was added to the flask and heated to decompose acetic anhydride that was not consumed by acetylation into acetic acid, and the amount of this acetic acid was neutralized with 0.5 mol / L potassium hydroxide aqueous solution. Measure with Separately, a blank is measured by the same operation as described above without putting a sample. Since the difference in the number of moles between the above two measurements is the number of moles of acetic anhydride consumed for acetylation of the hydroxy groups of the sample (ie, the amount of hydroxy groups in the sample), this value is divided by the sample weight to give 1 g of sample. The amount of hydroxy groups per unit is obtained.

  The amount of the linker bound to the solid phase carrier is not particularly limited. However, if the amount of the linker is too small, the yield of the nucleic acid is decreased, whereas if the amount of the linker is too large, the purity of the obtained nucleic acid is reduced. The number of nucleotides of the obtained nucleic acid tends to be smaller than the desired number. Therefore, it is preferably in the range of 20 to 800 μmol / g, more preferably 25 to 500 μmol / g.

The shape of the porous solid phase carrier is not particularly limited, and may be any shape such as a flat plate shape, a particle shape, and a fiber shape. From the viewpoint that the container is difficult to break, it is preferably a porous synthetic polymer particle having a particle shape.
In the present specification, the term “particle” does not mean a strict spherical shape, but a fixed shape (for example, an approximately spherical shape such as an elliptical sphere, a polyhedral shape, a cylindrical shape, an atypical shape such as a confetti shape, etc. ).
The size (volume) of one porous synthetic polymer particle is not particularly limited, but if the average particle diameter measured by laser diffraction (scattering method) of the porous particle is smaller than 1 μm, the column is packed. When used, there is a problem that the back pressure becomes too high or the liquid feeding speed becomes slow. On the other hand, if the average particle size is larger than 1000 μm, the gap between the carrier particles becomes large when packed in the column, It becomes difficult to efficiently load carrier particles into a fixed volume column. Therefore, it is preferably 1 to 1000 μm, more preferably 5 to 500 μm, and still more preferably 10 to 200 μm.

The specific surface area measured by the multipoint BET method of the porous synthetic polymer particles is not particularly limited, but if the specific surface area is less than 0.1 m ² / g, the degree of swelling in an organic solvent will be low, so that the synthesis reaction will be reduced. On the other hand, when it is larger than 500 m ² / g, the pore diameter becomes small, so that the synthesis reaction tends to hardly occur. Therefore, it is preferably 0.1 to 500 m ² / g, more preferably 10 to 300 m ² / g, and still more preferably 50 to ²⁰⁰ m ² / g.

  Further, the average pore diameter measured by the mercury intrusion method of the porous synthetic polymer particles is not particularly limited, but if the pore diameter is too small, the synthetic reaction field becomes small and the desired reaction hardly occurs, or the nucleotide length Tends to be less than the desired number, and on the other hand, if the pore size is too large, the chance of contact between the hydroxyl groups on the surface of the polymer particles as the reaction field and the substance involved in the reaction tends to decrease, and the yield tends to decrease. There is. Therefore, it is preferably 1 to 200 nm, more preferably 5 to 100 nm, and still more preferably 20 to 70 nm.

  In a preferred embodiment of the solid phase carrier of the present invention, Sp is a low-swelling crosslinked polystyrene particle commercially available as NittoPhase (registered trademark) (manufactured by Nitto Denko Corporation). The solid-phase nucleic acid synthesis method using NittoPhase (registered trademark) is preferably used because it has a small peak area due to impurities and guarantees high yield and high purity in a wide range of scales from a laboratory scale to a large-scale synthesis system.

Method for Producing Nucleic Acid Solid Phase Synthesis Carrier of the Present Invention The method for producing the nucleic acid solid phase synthesis carrier of the present invention is not particularly limited, and for example, it can be produced by the following method.

A diepoxynaphthalene derivative is synthesized by reacting alkyne (for example, acetylene, dimethylacetylene) with furan or the like. Next, this derivative is oxidized using osmium tetroxide as a catalyst and reacted with 4,4′-dimethoxytrityl chloride and the like to protect a part of the hydroxy group with a dimethoxytrityl group (DMTr group). Subsequently, succinic anhydride is reacted with this compound together with dichloromethane and triethylamine, and a succinyl linker moiety is bound to the remaining hydroxy group, and this is bound to a solid phase carrier having —OH group or —NH ₂ group. The carrier for nucleic acid solid phase synthesis of the present invention can be obtained.

Nucleic Acid Synthesis Method Using Nucleic Acid Solid-Phase Synthesis Carrier of the Present Invention Nucleic acid synthesis using the nucleic acid solid-phase synthesis carrier of the present invention can be carried out by various known synthesis methods using an automatic nucleic acid synthesizer. In the present specification, the “nucleic acid synthesis reaction” means an elongation reaction of nucleotides constituting a nucleic acid. That is, an elongated oligonucleotide is obtained by sequentially binding nucleotides to nucleosides, nucleotides or oligonucleotides bound on a solid support.
Examples of the nucleic acid synthesis reaction include an H-phosphonate method, a phosphoester method, a solid phase phosphoramidite method, and the like. Among them, a high purity nucleic acid is obtained because of high nucleic acid synthesis ability. The phosphoramidite method is preferred.

Preferred embodiments of the nucleic acid synthesis reaction by the solid phase phosphoramidite method include, for example, the following steps (a) a step of placing the nucleic acid solid phase synthesis carrier of the present invention in a reaction column of an automatic nucleic acid synthesizer;
(B) a step of flowing an acid such as a dichloroacetic acid solution through the reaction column to deprotect and wash the protective group of the hydroxymethyl group;
(C) The steps of coupling the nucleoside phosphoramidite corresponding to the 3 ′ end activated by tetrazole or the like to the hydroxymethyl group, capping the unreacted hydroxy group, and oxidizing the phosphite are sequentially performed. And repeating this series of steps until the target sequence is achieved;
(D) After completion of the synthesis step in the apparatus, a step of immersing the nucleic acid solid phase synthesis carrier in ammonia water or the like to cut the ligated portion to obtain the target nucleic acid;
The method comprised from these is mentioned.

  By the production method described above, a chemical reaction represented by the following formula occurs, and a nucleic acid having a hydroxy group at the 3 ′ end is generated. That is, in the nucleic acid synthesis method using the nucleic acid solid phase synthesis carrier (A) shown in Example 2 as a preferred embodiment of the present invention, a chemical reaction represented by the following formula occurs, and the 3 ′ end is hydroxylated. A nucleic acid having a group is produced.

[Wherein sphere represents a solid phase carrier, Ac represents an acetyl group, and Nuc represents a nucleic acid. ]

  Moreover, in the nucleic acid synthesis method using the nucleic acid solid phase synthesis carriers (B) and (C) of the present invention shown in Examples 4 and 6 as a preferred embodiment of the present invention, the chemistry represented by the following formula: A reaction occurs, and a nucleic acid having a hydroxy group at the 3 ′ end is produced.

[In the formula, a sphere represents a solid phase carrier, and Nuc represents a nucleic acid. ]

  As the activator used in the nucleic acid production method of the present invention, 1H-tetrazole, 4,5-dicyanoimidazole, 5-ethylthio-1H-tetrazole, benzimidazolium triflate (BIT), N-phenylbenzimidazolium triflate, imidazo Examples include, but are not limited to, triflate (IMT), N-PhIMP, 5-nitrobenzimidazolium triflate, triazolium triflate, 1-hydroxybenzotriazole (HOBT), or N- (cyanomethyl) pyrrolidinium tetrafluoroborate. It is not limited to.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples described below.

Example 1
Preparation of Nucleic Acid Solid Phase Synthesis Support (A) Acetylenedicarboxylic acid dimethyl ester and furan were added to a Schlenk type reaction tube and reacted at room temperature for 29 days. The yield of this reaction was 22%. The product was oxidized using osmium tetroxide as the catalyst. Next, 4,4′-dimethoxytrityl chloride and the like were reacted to protect some hydroxy groups with DMTr groups. Thereafter, a porous polystyrene-based solid phase carrier having a hydroxy group (Nitto Denko Co., Ltd., NittoPhase (registered trademark)) into which succinyl groups are introduced is dispersed in acetonitrile, and the above compound, HBTU, N, N-diisopropyl is dispersed. Ethylamine was added and reacted at 28 ° C. for 23 hours, and the compound was supported on a solid phase carrier. Subsequently, 4-dimethylaminopyridine, acetonitrile, ethanol, diisopropylethylamine and HBTU were added and reacted at 28 ° C. for 20 hours to cap the unreacted carboxy group, and acetic anhydride, N-methylimidazole, pyridine, 4- Dimethylaminopyridine and acetonitrile were added and reacted at 28 ° C. for 22 hours to cap the unreacted hydroxy group.

[In the formula, a sphere represents a solid phase carrier, and Ac represents an acetyl group. ]

The solid phase carrier (A) for nucleic acid synthesis of the present invention represented by
The amount of the linker of the present invention obtained as described above bound to the solid phase carrier was 41 μmol / g.

Example 2
Synthesis of DNA 20mer using carrier for nucleic acid solid phase synthesis (A) 24.3 mg of solid phase carrier for nucleic acid synthesis (A) of the present invention prepared in Example 1 was packed in a reaction column, and DNA / RNA automatic synthesizer Using ABI3400 (manufactured by Applied Biosystems), a DNA oligonucleotide of 20mer (5′-ATACCGATT AAGGCGAAGTT-3 ′: SEQ ID NO: 1) was synthesized by DMT-on (method without removing 5′-end protecting group) (synthesis scale) 1 μmol). After synthesis, the solid phase carrier to which the DNA oligonucleotide is bound is immersed in a 30% ammonia water / ethanol (3: 1) mixed solution at 55 ° C. for 15 hours to excise the DNA oligonucleotide from the solid phase carrier. It was.

Comparative Example 1
Cleavage into a commercially available solid phase carrier NittoPhase (registered trademark) (manufactured by Nitto Denko Corporation) in the same manner as in Synthesis Example 1 of DNA20mer using a solid phase synthesis carrier to which DMT-dT-3′-succinate was bound. The sexual linker DMT-dT-3′-succinate (from Beijing OM Chemicals) was conjugated. The amount of the compound bound to the solid phase carrier was 42 μmol / g. 23.8 mg of this solid phase carrier was packed into a reaction column, and a 20-mer (5′-ATACCGATTAAGCGAAGTT-3 ′: SEQ ID NO: 1) DNA oligonucleotide was added to DMT-on (5′-end protecting group) in the same manner as in Example 2. (Method not to remove) (synthesis scale 1 μmol). After synthesis, the DNA oligonucleotide was excised from the solid support.

Experimental example 1
The DNA oligonucleotide solutions obtained in Example 2 and Comparative Example 1 were measured by high performance liquid chromatography (HPLC) (measuring conditions: column; Waters XBridge OST C18 2.5 μm 50 × 4.6 mm, UV Detection; 260 nm, Buffer A; 100 mM HFIP / 7 mM TEA in Water, pH 8.0, Buffer B; Methanol, temperature; 30 ° C.). The respective HPLC charts are shown in FIGS. 1 (a) and 1 (b).
In addition, LC-MS analysis was performed on the DNA oligonucleotide solutions obtained in Example 2 and Comparative Example 1. (Measurement conditions: column; Waters XBridge OST C18 2.5 μm 50 × 4.6 mm, UV detection; 254 nm, Buffer A; HFIP / 7 mM TEA in Water, pH 8.0, Buffer B; methanol, temperature; 30 ° C.).
As a result, it was confirmed that the main peak of the DNA oligonucleotide prepared in Example 2 was a DNA oligonucleotide 20mer having a hydroxy group at the 3 ′ end (molecular weight (measured value); 6439). On the other hand, the main peak of the DNA oligonucleotide prepared in Comparative Example 1 was also confirmed to be a DNA oligonucleotide 20mer having a hydroxy group at the 3 ′ end (molecular weight (measured value); 6439).

Example 3
Preparation of Nucleic Acid Solid Phase Synthesis Support (B) Acetylenedicarboxylic acid dimethyl ester and furan were added to a Schlenk type reaction tube and reacted at room temperature for 29 days. The yield of this reaction was 22%. The product was oxidized using osmium tetroxide as the catalyst. Next, 4,4′-dimethoxytrityl chloride and the like were reacted to protect some hydroxy groups with DMTr groups. Next, succinic anhydride or the like was added to introduce a succinyl group into the above compound. NittoPhase (registered trademark) (manufactured by Nitto Denko Corporation), which is a crosslinked polystyrene-based solid phase carrier having a hydroxy group, is dispersed in acetonitrile, and the above compound, HBTU, N, N-diisopropylethylamine is added, The reaction was carried out for a period of time and supported on a solid support. Thereafter, 4-dimethylaminopyridine, acetonitrile, ethanol, diisopropylethylamine and HBTU were added and reacted at 28 ° C. for 20 hours to cap the unreacted carboxy group, acetic anhydride, N-methylimidazole, pyridine, 4-dimethyl. Aminopyridine and acetonitrile were added and reacted at 28 ° C. for 22 hours to cap the unreacted hydroxy group.

[In the formula, a sphere represents a solid phase carrier. ]

The solid phase carrier (B) for nucleic acid synthesis of the present invention represented by
The amount of the linker of the present invention obtained as described above bound to the solid phase carrier was 141 μmol / g.

Example 4
Using a carrier for solid phase synthesis of nucleic acid (B), 7.1 mg of the solid phase carrier for nucleic acid synthesis (B) of the present invention prepared in Synthesis Example 3 of DNA20mer was packed in a reaction column, and an automatic DNA / RNA synthesizer , A DNA oligonucleotide of 20mer (5′-ATA CCG ATT AAG CGA AGT TT-3 ′: SEQ ID NO: 1) using ABI3400 (manufactured by Applied Biosystems) and DMT-on (method without removing 5′-end protecting group) ) (Synthesis scale 1 μmol). After the synthesis, the solid phase carrier to which the DNA oligonucleotide was bound was immersed in a 30% ammonia water / ethanol (3: 1) mixed solution at 55 ° C. for 15 hours to excise the DNA oligonucleotide from the solid phase carrier. .

Comparative Example 2
A solid phase to which DMT-dT-3′-succinate was bound in the same manner as in Comparative Example 1 of DNA 20mer using a carrier for solid phase synthesis to which DMT-dT-3′-succinate was bound. The carrier NittoPhase was prepared. 10.4 mg of this solid phase carrier was packed in a reaction column, and in the same manner as in Example 4, a 20mer (5′-ATA CCG ATT AAG CGA AGT TT-3 ′: SEQ ID NO: 1) DNA oligonucleotide was added to DMT-on. (Synthesis scale 1 μmol). After synthesis, the DNA oligonucleotide was excised from the solid support.

Experimental example 2
The DNA oligonucleotide solutions obtained in Example 4 and Comparative Example 2 were subjected to HPLC measurement (measurement conditions: column; XBridge OST C18 2.5 μm 50 × 4.6 mm (manufactured by Waters), UV detection; 260 nm, Buffer A; 100 mM HFIP / 7 mM TEA / water (pH 8.0), Buffer B; methanol, temperature; 30 ° C.). Each HPLC chart is shown in FIGS. 2 (a) and 2 (b).
As a result, the main peak of the DNA oligonucleotide prepared in Example 4 had the same detection time as that in Example 2, and thus was confirmed to be a DNA oligonucleotide 20mer having a hydroxy group at the 3 ′ end. On the other hand, since the main peak of the DNA oligonucleotide prepared in Comparative Example 2 has the same detection time as Comparative Example 1, it was confirmed that the DNA oligonucleotide was a 20-mer DNA oligonucleotide having a hydroxy group at the 3 ′ end.

Example 5
Preparation of Nucleic Acid Solid Phase Synthesis Support (C) Acetylenedicarboxylic acid, tetrahydrofuran and furan were added to a Schlenk type reaction tube and reacted at room temperature for 18 days. The yield of this reaction was 67%. The resulting dicarboxylic acid was mixed with dimethyl sulfate, potassium carbonate, and acetone and refluxed to change to the corresponding dimethyl ester. The product was oxidized using osmium tetroxide as the catalyst. Next, 4,4′-dimethoxytrityl chloride and the like were reacted to protect some hydroxy groups with DMTr groups, followed by column chromatography purification. Next, succinic anhydride or the like was added to introduce a succinyl group into the above compound. NittoPhase (registered trademark) (manufactured by Nitto Denko Corporation), which is a crosslinked polystyrene-based solid phase carrier having a hydroxy group, is dispersed in acetonitrile, and the above compound, HBTU, N, N-diisopropylethylamine is added, The reaction was carried out for a period of time and supported on a solid support. Thereafter, 4-dimethylaminopyridine, acetonitrile, ethanol, diisopropylethylamine and HBTU were added and reacted at 28 ° C. for 20 hours to cap the unreacted carboxy group, acetic anhydride, N-methylimidazole, pyridine, 4-dimethyl. Aminopyridine and acetonitrile were added and reacted at 28 ° C. for 22 hours to cap the unreacted hydroxy group.

[In the formula, a sphere represents a solid phase carrier. ]

The solid phase carrier (C) for nucleic acid synthesis of the present invention represented by
The amount of the linker of the present invention obtained as described above bound to the solid phase carrier was 62 μmol / g.

Example 6
Synthesis of DNA 20mer using carrier for solid phase synthesis of nucleic acid (C) 16.1 mg of solid phase carrier for nucleic acid synthesis (C) of the present invention prepared in Example 5 was packed in a reaction column, and an automatic DNA / RNA synthesizer , A DNA oligonucleotide of 20mer (5′-ATA CCG ATT AAG CGA AGT TT-3 ′: SEQ ID NO: 1) using ABI3400 (manufactured by Applied Biosystems) and DMT-on (method without removing 5′-end protecting group) ) (Synthesis scale 1 μmol). After the synthesis, the solid phase carrier to which the DNA oligonucleotide was bound was immersed in a 30% ammonia water / ethanol (3: 1) mixed solution at 55 ° C. for 15 hours to excise the DNA oligonucleotide from the solid phase carrier. .

Comparative Example 3
Synthesis of DNA 20mer using carrier for solid phase synthesis coupled with DMT-dT-3'-succinate 10.4 mg of solid phase carrier coupled with DMT-dT-3'-succinate prepared in Comparative Example 2 was packed in a reaction column. Then, in the same manner as in Example 4, a 20mer (5′-ATA CCG ATT AAG CGA AGT TT-3 ′: SEQ ID NO: 1) DNA oligonucleotide was synthesized with DMT-on (synthesis scale: 1 μmol). After synthesis, the DNA oligonucleotide was excised from the solid support.

Experimental example 3
The DNA oligonucleotide solutions obtained in Example 6 and Comparative Example 3 were subjected to HPLC measurement (measurement conditions: column; XBridge OST C18 2.5 μm 50 × 4.6 mm (manufactured by Waters), UV detection; 260 nm, Buffer A; 100 mM HFIP / 7 mM TEA / water (pH 8.0), Buffer B; methanol, temperature; 30 ° C.). The respective HPLC charts are shown in FIGS. 3 (a) and 3 (b).
As a result, the main peak of the DNA oligonucleotide prepared in Example 6 was the same as that in Example 2, and thus was confirmed to be a DNA oligonucleotide 20mer having a hydroxy group at the 3 ′ end. On the other hand, since the main peak of the DNA oligonucleotide prepared in Comparative Example 3 has the same detection time as Comparative Example 1, it was confirmed that it was a DNA oligonucleotide 20mer having a hydroxy group at the 3 ′ end.

  Table 1 summarizes the data obtained in Experimental Examples 1 to 3.

  In recent years, with the development of nucleic acid pharmaceuticals, development of more efficient and functional nucleic acid synthesis methods is desired. The solid phase carrier for nucleic acid synthesis of the present invention can be used for nucleic acid synthesis having various modified sites in addition to normal nucleic acid synthesis. In addition, nucleic acids having a hydroxy group at the 3 ′ end automatically synthesized using the solid phase carrier for nucleic acid synthesis of the present invention can be widely used regardless of the type of nucleic acid medicine such as antisense, aptamer, siRNA and the like. .

SEQ ID NO: 1 artificially synthesized oligo 20mer

Claims (8)

The following general formula

[Where,
X ¹ and X ² each independently represent a protecting group for a hydroxy group selected from the group consisting of a trityl group, a monomethoxytrityl group and a dimethoxytrityl group ,
One of L ¹ and L ² represents a succinyl group, and the other represents a succinyl group or an acetyl group ,
R ¹ to R ⁶ are each independently hydrogen _{atom; C 1-6} optionally substituted alkoxy _{C 1-6} alkyl _{group; C 1-6} alkoxy _{group; C 1-7} acyl group; a mono Or a di-C _1-6 alkylamino group; or a halogen atom, or
R ² and R ⁵ together, together with the carbon atom to which they are attached, (1) an oxo group, (2) a C _1-6 alkyl group optionally substituted with a C _1-6 alkoxy group, And (3) a 3- to 8-membered carbocyclic or heterocyclic ring, each of which may be substituted with a substituent selected from a phenyl group which may be substituted with a C _1-6 alkoxy-carbonyl group. ]
In the indicated Ru, nucleic acid for solid phase synthesis linkers. The linker for nucleic acid solid phase synthesis according to claim 1, wherein at least one of L ¹ or L ² can be linked to a nucleic acid solid phase synthesis carrier having an amino group or a hydroxy group . The linker for nucleic acid solid phase synthesis according to claim 1 or 2, wherein X ¹ and X ² are dimethoxytrityl groups. The following general formula

[Where,
X ¹ and X ² each independently represent a protecting group for a hydroxy group selected from the group consisting of a trityl group, a monomethoxytrityl group and a dimethoxytrityl group ,
L ¹ represents a succinyl group,
L ² represents an acetyl group or an ethoxysuccinyl group ,
Sp represents a solid support,
R ¹ to R ⁶ are each independently hydrogen _{atom; C 1-6} optionally substituted alkoxy _{C 1-6} alkyl _{group; C 1-6} alkoxy _{group; C 1-7} acyl group; a mono Or a di-C _1-6 alkylamino group; or a halogen atom, or
R ² and R ⁵ together, together with the carbon atom to which they are attached, (1) an oxo group, (2) a C _1-6 alkyl group optionally substituted with a C _1-6 alkoxy group, And (3) a 3- to 8-membered carbocyclic or heterocyclic ring, each of which may be substituted with a substituent selected from a phenyl group which may be substituted with a C _1-6 alkoxy-carbonyl group. ]
A carrier for solid phase synthesis of nucleic acid having a structure represented by The carrier for nucleic acid solid phase synthesis according to claim 4, wherein X ¹ and X ² are dimethoxytrityl groups. The nucleic acid solid phase synthesis carrier according to claim 4 or 5 , wherein Sp is a solid phase carrier of porous synthetic polymer particles or porous glass particles. A method for producing a nucleic acid, comprising a step of performing a nucleic acid synthesis reaction on the nucleic acid solid phase synthesis carrier according to any one of claims 4 to 6 . The production method according to claim 7 , wherein the nucleic acid synthesis reaction is carried out by a solid phase phosphoramidite method.

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